Fourteen neo-clerodane diterpenoids isolated from Scutellaria altissima (Lamiaceae) were tested for insect antifeedant activity against Leptinotarsa decemlineata Say. Potato leaf disks treated with small amounts of the compounds (concentration 1000, 100, 10 ppm) resulted in good to very good antifeedant activity. Clerodin (1), scutecyprin (11) and 11-epi-scutecolumnin C (12) showed strong feeding inhibition at 1000 ppm and exhibited significant antifeedant activity at a concentration of 100 ppm. Activity was established by calculating the feeding ratio (FR) between the consumed areas of treated disks (CTD) and control disks (CCD). For comparison, FR50 values were determined as the FR at a CCD of 50%. Structural features of the compounds associated with the changes in activity and structure-antifeedant activity relationships are discussed. For the first time the anti-feedant activity has been evaluated of neo-clerodane diterpenoids with an unusual R-configuration of the carbon atom C-11.

Colorado potato beetle (CPB) adults and larvae devour leaves of potato and other solanaceous crops and weeds, and may quickly develop resistance to pesticides. With early detection of CPB damage, more options are available for precision integrated pest management, which reduces the amount of pesticides applied in a field. Remote sensing with small unmanned aircraft systems (sUAS) has potential for CPB detection because low flight altitudes allow image acquisition at very high spatial resolution. A five-band multispectral sensor and up-looking incident light sensor were mounted on a six-rotor sUAS, which was flown at altitudes of 60 and 30 m in June 2014. Plants went from visibly undamaged to having some damage in just 1 day. Whole-plot normalized difference vegetation index (NDVI) and the number of pixels classified as damaged (0.70 ≤ NDVI ≤0.80) were not correlated with visible CPB damage ranked from least to most. Area of CPB damage estimated using object-based image analysis was highly correlated to the visual ranking of damage. Furthermore, plant height calculated using structure-from-motion point clouds was related to CPB damage, but this method required extensive operator intervention for success. Object-based image analysis has potential for early detection based on high spatial resolution sUAS remote sensing.

Egg, larval, and adult life stages of Colorado potato beetle, Leptinotarsa decemlineata (Say), were observed feeding on or attached to a previously undocumented host plant belonging to the genus Chamaesaracha in eastern Colorado on July 2017. At one site, L. decemlineata were more abundant on Chamaesaracha sp. than the accepted ancestral host plant, Solanum rostratum (Dunal). While future studies should confirm the ancestral status of the observed L. decemlineata and suitability of Chamaesaracha sp. for completion of development, our observations suggest a need for further characterization of the ancestral host range of L. decemlineata.

Theory on condition-dependent risk-taking indicates that when prey are in poor condition, their anti-predator responses should be weak. However, variation in responses resulting from differences in condition is generally considered an incidental by-product of organisms living in a heterogeneous environment. Using Leptinotarsa decemlineata beetles and stinkbug (Podisus maculiventris) predators, we hypothesised that in response to predation risk, parents improve larval nutritional condition and expression of anti-predator responses by promoting intraclutch cannibalism. We showed that mothers experiencing predation risk increase production of unviable trophic eggs, which assures provisioning of an egg meal to the newly hatched offspring. Next, we experimentally demonstrated that egg cannibalism reduces L. decemlineata vulnerability to predation by improving larval nutritional condition and expression of anti-predator responses. Intraclutch cannibalism in herbivorous insects might be a ubiquitous strategy, aimed to overcome the dual challenge of feeding on protein-limited diets while living under constant predation threat.

Herbivorous insects use complex protease complements to process plant proteins, useful to adjust their digestive functions to the plant diet and to elude the antidigestive effects of dietary protease inhibitors. We here assessed whether basic profiles and diet-related adjustments of the midgut protease complement may vary among populations of the insect herbivore Colorado potato beetle (Leptinotarsa decemlineata). Two laboratory colonies of this insect were used as models, derived from insect samples collected in potato fields ∼1200 km distant from each other in North America. Synchronized 4th-instar larvae reared on potato were kept on this plant, or switched to tomato or eggplant, to compare their midgut cathepsin activities and content of intestain Cys proteases under different diet regimes. Cathepsin D activity, cathepsin L activity, cathepsin B activity and total intestain content shortly after larval molting on potato leaves were about two times lower in one population compared to the other. By comparison, cathepsin D activity, cathepsin B activity, total intestain content and relative abundance of the most prominent intestain families were similar in the two populations after three days regardless of the plant diet, unlike cathepsin L activity and less prominent intestain families showing population-associated variability. Variation in Cys protease profiles translated into the differential efficiency of a Cys protease inhibitor, tomato cystatin SlCYS8, to inhibit cathepsin L activity in midgut extracts of the two insect groups. Despite quantitative differences, SlCYS8 single variants engineered to strongly inhibit Cys proteases showed improved potency against cathepsin L activity of either population. These data suggest the feasibility of designing cystatins to control L. decemlineata that are effective against different populations of this insect. They underline, on the other hand, the practical relevance of considering natural variability of the protease complement among L. decemlineata target populations, eventually determinant in the success or failure of cystatin-based control strategies on a large-scale basis.

The main approach to pest control consists in the application of chemical insecticides. The efficacy of insecticides is reduced due to the development of resistance by pest populations. This is an especially important problem with the Colorado potato beetle. There are different strategies for the use of insecticides to slow the formation of resistance. Based on the results of long-term studies, we propose a hypothesis on delaying the development of resistance by applying insecticides at low doses. To test this hypothesis, we built predictive discrete genetic models of resistance in the Colorado potato beetle populations. The model based on the classical equations of population genetics has been supplemented by various factors. Calculations of the survival rates of individuals of the Colorado potato beetle were carried out taking into account the statistical regularities of the distribution of toxic substance after treatment by insecticides. We calculated the survival rates for different genotypes using a lognormal distribution after at least doubling the insecticide dose. The factor of differential mortality during winter was additionally introduced into the model. The use of phenetic markers of nonspecific resistance to environmental factors allowed us to compute the model with mediated intergenic interactions. Various hypotheses on the strategies of overcoming resistance have been tested using this model. The calculations demonstrated that use of insecticides at the minimum effective dose (low dose) leads to a slower increase in the proportion of resistant individuals in the populations of the Colorado potato beetle for two seasons. Resistance develops much more slowly following alternate treatment with insecticides from different chemical classes. The best strategy is through off-season treatment with lower doses of insecticides of different chemical classes.

Colorado potato beetle (CPB), Leptinotarsa decemlineata is a notorious pest of potato. Co-evolution with Solanaceae plants containing high levels of toxins (glycoalkaloids) helped this insect to develop an efficient detoxification system and resist almost every chemical insecticide introduced for its control. Even though the cross-resistance between plant allelochemicals and insecticides is well acknowledged, the underlying molecular mechanisms are not understood. Here, we investigated the molecular mechanisms involved in detoxification of potato plant allelochemicals and imidacloprid resistance in the field-collected CPB. Our results showed that the imidacloprid-resistant beetles employ metabolic detoxification of both potato plant allelochemicals and imidacloprid by upregulation of common cytochrome P450 genes. RNAi aided knockdown identified four cytochromes P450 genes (CYP6BJa/b, CYP6BJ1v1, CYP9Z25, and CYP9Z29) that are required for defense against both natural and synthetic chemicals. These P450 genes are regulated by the xenobiotic transcription factors Cap n Collar C, CncC and muscle aponeurosis fibromatosis, Maf. Studies on the CYP9Z25 promoter using the luciferase reporter assay identified two binding sites (i.e. GCAGAAT and GTACTGA) for CncC and Maf. Overall, these data showed that CPB employs the metabolic resistance mediated through xenobiotic transcription factors CncC and Maf to regulate multiple P450 genes and detoxify both imidacloprid and potato plant allelochemicals.

The responsiveness towards orally delivered dsRNA and the potency of a subsequent environmental RNA interference (RNAi) response strongly differs between different insect species. While some species are very sensitive to dsRNA delivery through the diet, others are not. The underlying reasons for this may vary, but degradation of dsRNA by nucleases in the gut lumen is believed to play a crucial role. The Colorado potato beetle, Leptinotarsa decemlineata, is a voracious defoliator of potato crops worldwide, and is currently under investigation for novel control methods based on dsRNA treatments. Here we describe the identification and characterization of two nuclease genes exclusively expressed in the gut of this pest species. Removal of nuclease activity in adults increased the sensitivity towards dsRNA and resulted in improved protection of potato plants. A similar strategy in the desert locust, Schistocerca gregaria, for which we show a far more potent nuclease activity in the gut juice, did however not lead to an improvement of the RNAi response. Possible reasons for this are discussed. Taken together, the present data confirm a negative effect of nucleases in the gut on the environmental RNAi response, and further suggest that interfering with this activity is a strategy worth pursuing for improving RNAi efficacy in insect pest control applications.

The speed of toxic action of an insecticide is an indicator for control efficacy and has considerable practical importance. For agricultural pest control, fast-acting is an important feature for an insecticide to consistently reduce the amount of feeding damage. Butene-fipronil is a novel compound obtained via the structural modification of fipronil. However, information about the toxicity and speed of toxic action is still limited. In the present paper, we compared the toxic feature of butene-fipronil with seven other insecticides, of which imidacloprid and abamectin are slow-acting insecticides, and acephate, endosulfan, methomyl, α-cypermethrin and spinosad are fast-acting insecticides. We found that the contact and stomach toxicities of butene-fipronil were among the highest ever estimated to Leptinotarsa decemlineata and Drosophila melanogaster. The speed of toxic action of butene-fipronil was determined using median lethal time (LT50) at a dose (concentration) equivalent to LD80 values. For L. decemlineata, the values for butene-fipronil, imidacloprid, abamectin, acephate, endosulfan, methomyl, cypermethrin and spinosad were calculated to be 39.9, 36.5, 37.5, 20.2, 22.4, 23.8, 16.4 and 23.1 h, respectively. Those for D. melanogaster were 29.8, 31.5, 29.4, 14.0, 20.3, 18.1, 13.5, and 20.1 h, respectively. ANOVA analysis showed that butene-fipronil, imidacloprid, abamectin had similar LT50 values, whereas acephate, endosulfan, methomyl, spinosad and cypermethrin had comparable LT50 values. Thus, butene-fipronil belongs to slow-acting insecticides. Our results provide more empirical information for butene-fipronil potential application.

The effect of essential oil (EO) from anise (Pimpinellia anisum) on the mortality of young larvae of Colorado potato beetles has been studied. In our bioassays, P. anisum EO significantly increased the mortality of the second instar larvae of L. decemlineata. Significantly different values of LD50 and LD90 were established for acute (LD50 = 1.76, and LD90 = 8.29) as well as chronic toxicity (LD50 = 0.45, and LD90 = 1.01). Decrease of both values over experimental period was evident, which showed that the larval mortality was slow and cumulative. The composition of EO used for biological experiments was also assessed. The main component detected in EO from P. anisum was anethole (79.87%), followed by anisaldehyde (7.74%), estragole (5.88%) and β-linalool (1.07%). Within five days, residual concentration of EO decreased from 3.87 mg/g of dry weight immediately after foliar applications to 0.9 mg per g of dry weight. The effect of this slow evaporation could be explained by dominant presence of anethole or by the type of formulation and the addition of oil and tween. Results of our study demonstrate that EO from P. anisum has insecticidal properties that may lead to the development of new organic products for the control of Colorado potato beetles.